Abstracts of papers on haemophilia from other journals

The progression of HCV-associated liver disease in a cohort of haemophilic patients. Telfer P, Sabin C, Devereux H, Scott F, Dusheiko G, Lee C.
Increasing hepatitis C virus RNA levels in hemophiliacs: relationship to human immunodeficiency virus infection and liver disease. Eyster E, Fried MW, Di Bisceglie AM, Goedert JJ.
Risks of immunodeficiency, AIDS, and death related to purity of factor VIII concentrate. Goedert JJ, Cohen AR, Kessler CM, Eichinger S, Seremtis SV, Rabkin CS, Yellin FJ, Rosenberg PS, Aledort LM.
Synoviorthesis with colloidal 32P chronic phosphate for the treatment of hemophilic arthropathy. Rivard G-E, Girard M, Belanger R, Jutras M, Guay J-P, Marton D.
Continuous infusion of monoclonal antibodypurified factor VIII: rational approach to serious hemorrhage in patients with allo-/autoantibodies to factor VIII. Gordon EM, Al-Batniji F, Goldsmith JC.
Monoclonal purified F VIII for continuous infusion: stability, microbiological safety and clinical experience. Schulman S, Varon D, Keller N, Gitel S, Martinowitz U.     

Management of inherited von Willebrand disease.

von Willebrand disease is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor. The diagnosis is based on measurements of plasma and platelet von Willebrand factor, the ability of von Willebrand factor to interact with its platelet receptor and the analysis of the multimeric composition of von Willebrand factor. Owing to the heterogeneity of von Willebrand factor defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate therapy. The aim of treatment is to correct the dual defects of haemostasis, i.e. abnormal coagulation, expressed by a low level of factor VIII, and abnormal platelet adhesion, expressed by a prolonged bleeding time. Desmopressin is the treatment of choice in patients with type I von Willebrand disease, who account for approximately 70% of cases, because it corrects the factor VIII/von Willebrand factor level and the prolonged bleeding time in most of these patients. In type 3 and in the majority of type 2 von Willebrand disease patients, desmopressin is not effective and it is necessary to resort to plasma concentrates containing factor VIII and von Willebrand factor. Treated with virucidal methods, these concentrates are effective and currently safe, but they do not always correct the bleeding time defect. Platelet concentrates or desmopressin can be used as adjunctive treatments when a poor correction of the bleeding time after concentrates is associated with continued bleeding. Studies are in progress, first to better characterize patients with type I von Willebrand disease and to determine their response to desmopressin, and second, to evaluate the pharmacokinetics of factor VIII following factor VIII/von Willebrand factor concentrates and to establish the indication for concentrates.     

Further evidence that the residual vWf:Ag in porcine FVIII:C induces human platelet aggregation

Porcine or bovine factor VIII concentrates (FVIII:C) have been used during the past 3 decades to control bleeding in patients who have developed antibodies to human factor VIII. Since current preparations of animal FVIII:C are not known to transmit infectious agents such as hepatitis or human immunodeficiency virus, they are of potential therapeutic interest. A purified porcine FVIII:C (Hyate:C) is now widely used as an alternative to human FVIII:C in patients with inhibitor. Unlike earlier preparations of porcine FVIII:C, thrombocytopaenia is rare with the current preparation. Nonetheless, it causes the aggregation of human platelets in vitro. Our aim was to identify precisely the plasma factor which induces platelet aggregation. The effects of commercial porcine FVIII:C, porcine fibrinogen, porcine fibronectin and the corresponding preparations from human origin on platelet aggregation were studied. Platelet aggregation was quantified by measuring the fall in single platelet count in human whole blood. Of these preparations, only porcine FVIII:C (0.1-1 U/ml) and porcine fibrinogen (80-600 micrograms/ml) induced a fall in single platelet count of up to 85% due to aggregation. The extent of aggregation was directly proportional to the amount (0.007-0.1 U/ml test aliquot) of residual von Willebrand factor antigen (vWf:Ag) in the preparations. A monoclonal antibody to vWf:Ag inhibited the aggregation. We believe that the aggregation of human platelets induced in vitro by porcine FVIII:C is mediated by vWf:Ag which also may be responsible for thrombocytopaenia reported following administration of porcine FVIII:C in vivo.     

The impact of hepatitis C antibody screening of source plasma donors on hepatitis C virus RNA in factor VIII concentrates

Summary. We have used the polymerase chain reaction technique for the detection of hepatitis C RNA in nine different plasma-derived factor VllI concentrates and in two factor IX concentrates. Four concentrates were investigated both prior to and after the introduction of donor screening for hepatitis C antibodies. A negative reaction was consistently found in the ultra-pure factor VIII concentrates Octonativ-M (Pharmacia) and Hemofil M (Baxter), both prepared by affinity purification with factor VI1I:C monoclonal antibodies and virus inacti- vated hy solvent/detergent procedures, as well as in both the low-purity factor IX concentrates. I f produced from unscreened plasma, the other factor VIII concentrates manifested positive reactions irrespective of preparation procedure and type of virus inactivation or the temperature at which it was performed. We conclude that the preparation procedure of clotting factor concentrates, rather than type of virus inactivation, determines the degree of contamination by hepatitis C virus RNA, and that screening of source plasma seems effective in removing hepatitis C RNA from the final product as determined with a sensitive PCR method. I t is important to stress that the presence of viral RNA does not necessarily imply clinical infectivity.     

Von Willebrand factor contaminating porcine factor VIII concentrate (Hyate: C) causes platelet aggregation

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.     

Variation in factor VIII inhibitor reactivity with different commercial factor VIII preparations

Summary. During treatment of a haemophilia A patient with a high-responding inhibitor against factor VIII coagulant activity (VIII:C), we observed a difference in recovery of VIII:C depending upon which factor concentrate was infused. Inhibitor plasma samples or IgG fraction from seven patients were tested against a panel of seven different commercially available factor VIII concentrates of which five were plasma-derived and two recombinant. In two of the plasma samples, inhibitor titres manifested a wide range of values depending upon which concentrate was used in the test system. Thus, inhibitor neutralization was less and VIII:C recovery greater when factor VIII concentrates containing large amounts of von Willebrand factor were used than when highly purified concentrates containing no von Willebrand factor or only trace amounts were used. In both of these two patients the inhibitor was directed against the light chain of factor VIII, and it is possible that the epitope of the light chain with which the inhibitor reacts is partly blocked by the von Willebrand factor.
We conclude that inhibitors may differ in their reactivity with factor VIII molecules contained in clotting factor concentrates, and that there is factor VIII epitope variation between different concentrates. These findings have implications for the selection of concentrates for the treatment of inhibitor patients and the haemostatic effect may be improved if a concentrate giving the lowest inhibitor titre is chosen. Thus, in vitro testing of inhibitor reactivity with a panel of concentrates is recommended when treatment of inhibitor patients with factor VIII concentrates is considered.     

Increased factor VIII/vWf levels in patients with reduced platelet number

Summary Factor VIII/von Willebrand factor (VIII/vWf) related properties were studied in twenty six patients with thrombocytopenia. Fifteen patients were affected by idiophatic thrombocytopenic purpura (ITP) and 11 patients by thrombocytopenia of a different nature or non-ITP (n-ITP). All patients showed an enhancement of platelet associated IgG (PAIgG). A significant increase of factor VIII ristocetin cofactor (VIII R: RCoF) and factor VIII related antigen (VIII R: Ag) was found in ITP patients while normal values were observed for factor VIII coagulant (VIII: C). All factor VIII/vWf components, on the contrary, were increased in n-ITP group with a prevalence of VIII R: RCoF as observed in ITP group even though with lower mean values. Multimeric analysis of VIII/vWf demonstrated a higher concentration of all multimeric components, with major representation of higher molecular weight multimers (HMWM) in patients of both groups.Two patients were studied before and after improvement in platelet count. A decrease of vWf related properties (VIII R : RCoF and VIII R : Ag) concomitant with the increase in platelet count was found. In n-ITP patients a statistical correlation between VIII R : RCoF and PAIgG was also observed while no correlation was found between other factor VIII/vWf components and PAIgG both in ITP and n-ITP patients.     

IgA inhibitor to factor VIII/von Willebrand factor

A 60-year-old Black female presented with a haemorrhagic diathesis and an acquired factor VIII/von Willebrand factor (VIII/vWf) inhibitor. This inhibitor was classified as an IgA immunoglobulin and was active not only against factor VIII coagulant (VIII:C) activity but also against plasma von Willebrand factor (vWf). The purified IgA also interacted with normal platelets to inhibit ristocetin-induced platelet aggregation (RIPA). In contrast, studies with haemophilia A plasma and platelets revealed that the inhibitor did not react significantly with these plasmas or platelets. The significant differences in the inhibition of vWf assay both of the plasma and the platelets of the haemophilia A patients suggests that part of the haemorrhagic diathesis may be related not only to the inhibition of VIII:C but also to interference with platelet function. In addition, these studies suggest that there may be significant differences in the factor VIII-related antigen (VIII R:Ag) on platelets in haemophilia A patients compared to normal.     

Factor VIII:C and VIII:CAg Response in Patients with Haemophilia A and von Willebrand's Disease after Administration of Different Factor VIII Concentrates or Plasma

S ummary . Factor VIII procoagulant activity (VIII:C) and factor VIII procoagulant antigen (VIII:CAg) were studied in seven patients with haemophilia A after administration of three different factor VIII concentrates or plasma. The in vivo recovery of VIII:CAg was less than that of VIII:C and the disappearance rate of VIII:CAg was much higher either when concentrates or plasma were given. The half-life of VIII:C was thus about 12 h but of VIII:CAg only about 3 h or less. Six patients with von Willebrand's disease were studied after administration of AHF- Kabi. In contrast to haemophilia A the discrepancy between VIII:C and VIII:CAg disappearance rates was not present in von Willebrand's disease, since both VIII:C and VIII:CAg showed a typical progressive increase. We conclude that factor VIII:C given to haemophilia patients does not behave like native VIII:C, not even when fresh plasma is used. Patients with von Willebrand's disease are capable of forming a normal VIII:C when appropriately stimulated.     

Procoagulant versus anticoagulant factors in cirrhotic patients

Background and study aimLiver cirrhosis leads to decreased production of clotting factors that are generally all produced in the liver except factor VIII and von Willebrand factor. However, cirrhotic patients are not protected from thrombosis. The present study aimed to assess the procoagulant and anticoagulant factors in cirrhotic patients with and without bleeding and/or thrombotic events.Patients and methodsA total of 102 adult subjects were enroled: 51 cirrhotic patients and 51 healthy controls. After full history taking with special attention given to thromboembolic and haemorrhagic events, platelet count, serum albumin, bilirubin, international normalised ratio (INR), PT, partial thromboplastin time (PTT), hepatitis B surface antigen (HBsAg), hepatitis B core (HBc) antibodies, hepatitis C virus (HCV) antibodies, factor VIII, protein C, Protac-induced coagulation inhibition percentage (PICI%) assay and abdominal ultrasound were performed for patients and controls. Upper gastrointestinal endoscopy was conducted for the patients.ResultsCompared with control subjects, factor VIII and factor VIII/protein C were significantly higher, while protein C and PICI% were significantly lower among patients.ConclusionPatients with liver cirrhosis may have a tendency for bleeding or thrombosis according to the balance of coagulant and anticoagulant status. PICI%, the assay that evaluated the functionality of the protein C anticoagulant system, was significantly lower in patients compared to control subjects. Accordingly, low PICI% and high factor VIII/protein C ratio can be taken as an index of hypercoagulability in cirrhotic patients.     

Circulating immune complexes in haemophilia and von Willebrand's disease

Sera from 63 patients with haemophilia A, 21 with haemophilia B and 29 with von Willebrand's disease were screened for the presence of circulating immune complexes (CICs), serological markers of hepatitis A and B virus, autoantibodies and factor VIII or factor IX inhibitors. CICs were detected by the 125J Clq binding assay (ClqBA), the solid phase conglutinin assay (KgBSP) and the solid phase Clq assay (ClqSP). The incidence of CICs detected by the ClqBA and the ClqSP methods in haemophiliacs and in von Willebrand patients was higher than that observed in normal subjects, while the prevalence of CICs detected by the KgBSP method was not. The presence of CICs was not correlated with patient age, severity of disease, presence of hepatitis B virus serological markers, abnormal liver function tests or factor VIII inhibitors. A significant connection was demonstrated between CICs detected by the ClqBA method and replacement therapy when the dose administered over 1 year was over 20 000 U of factor VIII or IX concentrates. The high proportion of CICs in von Willebrand's disease, not connected with the replacement therapy or the presence of serological markers of hepatitis virus, is in agreement with the possibility that immune complexes may be related to the disease itself and independent, at least in part, of exogenous agents.     

Acute Hepatitis Non-A, Non-B following Administration of Factor VIII Concentrates

A retrospective survey on clinical hepatitis in patients with bleeding disorders was performed. Nine episodes of hepatitis non-A, non-B occurred in 8 out of 20 patients (40%) with mild hemophilia A or von Willebrand's disease, who had been treated with commercial factor VIII concentrates. Only two episodes of hepatitis B occurred during the study period. The non-A, non-B attack rate after the first treatment was 40 % with factor VIII concentrate obtained from large plasma pools (=2,000 donors) including professional plasma donors as compared to 8 % after treatment with factor VIII concentrate obtained from smaller (100–250 donors) plasma pools from Scandinavian donors.     

Book Reviews

Book reviewed in this article: Self-assessment color review of clinical hematology. A. B. Mehta Inhibitors to coagulation factors. Advances in experimental medicine and biology, volume 386. L. M. Aledort, L. W. Hoyer, J. M. Lusher, H. M. Reisner, G. C. White II (Eds) Recent advances in haematology. M. K. Brenner, A. V. Hoffbrand (Eds)     

Pasteurized, monoclonal antibody factor VIII concentrate: establishing a new standard for purity and viral safety of plasma-derived concentrates.

A factor VIII concentrate (Monoclate-P) manufactured using a combination of pasteurization and immunoaffinity chromatography has been chosen to compare and contrast manufacturing aspects of plasma-derived factor VIII concentrates. Pasteurization is a virucidal method with a long safety record in clinical practice, while immuno-affinity chromatography selectively isolates and purifies the procoagulant protein of factor VIII, and partitions potential viral contaminants and nonessential proteins to the unbound fraction. The complete Monoclate-P production process reduces human immunodeficiency virus by > or = 10.5 log10, Sindbis (a model for hepatitis C virus) by > or = 6.5 log10, and murine encephalomyocarditis virus (a non-enveloped model virus) by 7.1 log10. The viral safety of Monoclate-P has been further demonstrated in clinical studies in patients not previously treated with blood or plasma-derived products. Additionally, the manufacture of Monoclate-P includes careful donor screening and plasma testing for antibodies to syphilis and human immunodeficiency, hepatitis B, and hepatitis C viruses to enhance source plasma safety. Combined with donor selection and plasma testing, multiple viral reduction steps effectively eliminate both lipid-enveloped viruses (e.g. human immunodeficiency, hepatitis B and C) and non-lipid-enveloped viruses (e.g. hepatitis A). In addition, polymerase chain reaction-based nucleic acid detection tests for hepatitis B and C viruses and for human immunodeficiency virus-1 have been introduced as part of an investigational new drug mechanism.     

Characteristics of Various Factor VIII Concentrates used in Treatment of Haemophilia A

Summary . Five different factor VIII concentrates, AHF-Kabi (fraction I-o), Krynativ-Kabi (freeze-dried cryoprecipitate), Hemofil-Hyland, AHF-profilate-Abbott, Kryobulin-Immuno, available in Sweden for treatment of haemophiliacs were compared with respect to in vivo recovery, half-life of factor VIII clotting activity (VIII:C) and in vitro properties. The parameters studied were VIII:C, factor VIII related antigen (VIIIR:AG), crossed immunoelectrophoresis, ristocetin co-factor activity (VIII: Rcof), fibrinogen content and factor XIII activity. AHF-Kabi had the lowest concentration of VIII:C. All the preparations had higher values for VIIIR:AG than for VIII:C. The quotient was highest for Hemofil, Krynativ-Kabi and Kryobulin and varied between 4 and 7. The lowest quotient, 1.3 to 4, was that of AHF-Kabi. The number of units of VIII: Rcof was almost the same as that of VIII:C. AHF-Kabi had the highest fibrinogen concentration and was the only preparation with high amounts of F XIII. In crossed immunoelectrophoresis AHF-Kabi showed a similar pattern to that of normal plasma. The other preparations had a different pattern suggesting less heterogenicity of the molecule. The in vivo recovery was about the same for all the concentrates. The disappearance rate of VIII:C after infusion of corresponding doses of the different concentrates was studied in six patients with severe haemophilia A who were not bleeding. AHF-Kabi had a half-life between 18 and 26 h which was substantially longer than any of the other preparations. The half-life of Hemofil, Krynativ-Kabi, AHF-Profilate and Kryobulin varied between 8 and 16 h. The concentrates were given to three patients with severe classical von Willebrand's disease. All the preparations caused an increase of VIII:C and VIIIR:AG as well as of VIII:Rcof, but only AHF-Kabi corrected the prolonged bleeding time. Knowledge of the properties of various VIII concentrates is of importance for the choice of treatment in haemophilia and von Willebrand's disease.     

Pregnancy and delivery in women with von Willebrand��s disease and different von Willebrand factor mutations

Background

Pregnancy in von Willebrand’s disease may carry a significant risk of bleeding. Information on changes in factor VIII and von Willebrand factor and pregnancy outcome in relation to von Willebrand factor gene mutations are very scanty.

Design and Methods

We examined biological response to desmopressin, changes in factor VIII and von Willebrand factor and pregnancy outcome in a cohort of 23 women with von Willebrand’s disease characterized at molecular level and prospectively followed during 2000–2007.

Results

Thirty-one pregnancies occurred during the study period. Remarkably, similar changes of factor VIII and von Willebrand factor were observed after desmopressin and during pregnancy in nine women with R854Q, R1374H, V1665E, V1822G and C2362F mutations. Women with von Willebrand’s disease and R1205H and C1130F mutations (17 pregnancies in 12 women) had only a slight increase of factor VIII and von Willebrand factor during pregnancy while their response to desmopressin was marked but short-lived. For these women, two to three desmopressin administrations within the first 48 hours were sufficient to successfully manage vaginal delivery. Two women with recessive von Willebrand’s disease due to compound heterozygosity for different gene mutations had a spontaneous, major increase in factor VIII while von Willebrand factor remained severely reduced. Desmopressin increased factor VIII and was clinically useful in the first case, while a factor VIII/von Willebrand factor concentrate was required in the second patient not responsive to the compound. Factor VIII/von Willebrand factor concentrate was also required for two women with type 2 A von Willebrand’s disease with V1665E mutations who had no von Willebrand factor activity change during pregnancy. In one of them, delayed bleeding occurred 15 days later requiring treatment with Factor VIII/von Willebrand factor concentrate. No miscarriages or stillbirths occurred.

Conclusions

Close follow-up and detailed guidelines for the management of parturition have produced a very low rate of immediate and late bleeding complications in this setting. Desmopressin was effective and safe in preventing significant bleeding at delivery in most of these patients.     

Treatment of von Willebrand disease

Summary. von Willebrand disease is the most frequent of inherited bleeding disorders (1:100 affected individuals in the general population). The aim of treatment is to correct the dual defects of haemostasis, i.e., abnormal coagulation expressed by low levels of factor VIII and abnormal platelet adhesion expressed by a prolonged bleeding time. There are two main options available for the management of von Willebrand disease: desmopressin and transfusion therapy with blood products. Desmopressin is the treatment of choice in patients with type 1 von Willebrand disease, who account for approximately 80% of cases. This pharmacological compound raises endogenous factor VIII and von Willebrand factors and thereby corrects the intrinsic coagulation defect and the prolonged bleeding time in most type 1 patients. In type 3 and in the majority of type 2 patients desmopressin is not effective, and it is necessary to resort to plasma concentrates containing factor VIII and von Willebrand factor. Treated with virucidal methods, these concentrates are effective and currently safe, but the bleeding time defect is not always corrected by them. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the bleeding time after concentrates is associated with continued bleeding.     

4 cases of acquired Willebrand factor deficiency associated with monoclonal dysglobulinemia

Acquired von Willebrand syndrome is reported in four patients with monoclonal IgG: benign gammapathy in three cases, multiple myeloma in one case; to our knowledge, this last association has not been previously reported. Coagulation abnormalities included a borderline bleeding time, a low platelet retention on glass beads, decreased levels of factor VIII coagulant activity (VIII: C), factor VIII related-antigen (VIII R: Ag) and ristocetin induced agglutination cofactor (VIII R: RC). The late clinical onset, the negative family history and the immunological abnormality suggest an acquired von Willebrand syndrome. After cryoprecipitate infusion the patients did not show the expected rise and there was no secondary increment in factor VIII: C. Time-dependent inhibition of factor VIII R: RC and factor VIII: C was found in one case only and was associated with qualitative abnormality of factor VIII R: Ag demonstrated by crossed-immunoelectrophoresis. It was not possible to interpret this last test in the other cases, due to the very low level of factor VIII R: Ag. The factor VIII abnormalities might be related to the binding and/or destruction of factor VIII by a circulating antibody, or to the adsorption of this factor on the malignant lymphocytes.     

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.     

Inactivation of the AIDS-Causing Retrovirus and Other Human Viruses in Antihemophilic Plasma Protein Preparations by Pasteurization

Heat treatment at 60 degrees C for 10 h in solution (pasteurization) was introduced into the manufacturing process of antihemophilic cryoprecipitate (AHC) and factor VIII concentrates (F VIII) to reduce the risk of transmission of hepatitis to hemophiliacs. Since the acquired immunodeficiency syndrome (AIDS) may also be transmitted to hemophiliacs by antihemophilic plasma protein preparations, we have investigated inactivation of the AIDS virus HTLV III by pasteurization in AHC or F VIII and included in this study cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), poliovirus and vaccinia virus. Each of these viruses was efficiently inactivated by pasteurization although considerable differences were observed between the different viruses HTLV III was rapidly inactivated, becoming nondetectable within 30-60 min. Our findings indicate that pasteurized AHC or F VIII should have a high margin of safety regarding the transmission of AIDS or any other infectious disease caused by viruses such as those tested.